La Crosse virus (LACV), family Bunyaviridae, is a mosquito-borne virus recognized as a major cause of pediatric encephalitis in North America with 70-130 symptomatic cases each year. The virus was first identified as a human pathogen in 1960 after its isolation from a 4 year-old girl who suffered encephalitis and died in La Crosse, Wisconsin. The majority of LACV infections are mild and never reported, however, serologic studies estimate infection rates of 10-30100,000 in endemic areas. LAC encephalitis has become the most commonly reported pediatric arboviral encephalitis in the US with 70-130 symptomatic cases a year with severe sequelae. For these reasons we are interested in developing a vaccine to prevent this CNS disease. Sequence analysis of the complete LACV genomes of low-passage LACVhuman1960, LACVmosquito1978, and LACVhuman1978 strains and of biologically cloned derivatives of each strain, indicates that circulating LACVs are genetically stable over time and geographic distance with 99.6-100%, 98.9-100%, 97.8-99.6%, and 99.2-99.7% amino acid identity for N, NsS, M polyprotein, and L proteins respectively. We identified 5 amino acid differences in the RNA polymerase and 4 nucleotide differences in the non-coding region of the L segment specific to the human virus isolates, which may result in altered disease outcomes. All three wild type viruses had similar in vitro growth kinetics and phenotypes in mosquito C636 and Vero cells, and similar levels of neurovirulence and neuroinvasiveness in Swiss Webster mice. The biologically cloned derivative of LACVhuman1960 was significantly less neuroinvasive than its uncloned parent and differed in sequence at one amino acid position in the GN glycoprotein, identifying this residue as an attenuating mutation.[unreadable] [unreadable] Much research this past year has been done to create murine and simian LACV infection models suitable for pathogenesis and vaccine safetyefficacy studies. As an initial step in vaccine development, we sought to characterize the LACV wild-type phenotype in vivo, we inoculated 3-week old Swiss Webster mice with either 1 or 100 LD50 of virus. For six consecutive days, 20 tissues were individually collected from each mouse and processed for virus titration or histopathology studies. Following inoculation, low viral titers were found in abdominal tissues near the inoculation site. By day 2 virus was detected in tissues distal to the inoculation site, including the nasal turbinates and brain. Throughout the study, there was a strong correlation between high viral titer in the nasal turbinates and subsequent dissemination of the virus into CNS tissue. Surprisingly, this mosquito-vectored virus can easily infect mice intranasally resulting in clinical disease. Intramuscular or subcutaneous inoculation of seronegative rhesus macaques leads to a subclinical infection with no detectable viremia, however these monkeys developed strong neutralizing antibody responses suggesting this will be a useful model for future studies on the immunogenicity of vaccine candidates.[unreadable] [unreadable] A reverse genetics system is being developed to permit the introduction of defined attenuating mutations into the genome of this virus. A mutant with a knock out of the NS-s coding region has been isolated that will form the basis for introduction of mutations that restrict replication in human neuronal tissue.